The main focus of the laboratory in 2004-5 was in elucidating structural mechanisms of lipid and phophoinositide signaling and protein trafficking, with an emphasis on mechanisms relevant to AIDS and type II diabetes and on fundamental mechanisms important to eukaryotic cell biology. Monoubiquitin and trafficking 1. Structure of the GGA GAT domain:ubiquitin complex The Golgi-localized, gamma adaptin-ear-containing, Arf-binding (GGA) proteins are clathrin adaptors that mediate the sorting of transmembrane cargo molecules at the trans-Golgi network and endosomes. Cargo proteins can be directed into the GGA pathway by at least two different types of sorting signals: acidic cluster-dileucine motifs, and covalent modification by ubiquitin. The latter modification is recognized by the GGAs through binding to their GAT domain. Here we report the crystal structure of the GAT domain of human GGA3 in a 1:1 complex with ubiquitin at 2.8 ? resolution. Ubiquitin binds to a hydrophobic and acidic patch on helices 1 and 2 of the GAT three-helix bundle that includes Asn-223, Leu-227, Glu-230, Met-231, Asp-244, Glu-246, Leu-247, Glu-250, and Leu-251. The GAT-binding surface on ubiquitin is a hydrophobic patch centered on Ile-44 that is also responsible for binding most other ubiquitin effectors. The ubiquitin-binding site observed in the crystal is distinct from the Rabaptin-5-binding site on helices 2 and 3 of the GAT domain. Mutational analysis and modeling of the ubiquitin:Rabaptin-5:GAT ternary complex indicates that ubiquitin and Rabaptin-5 can bind to the GAT domain at two different sites without any steric conflict. This highlights the GAT domain as a hub for interactions with multiple partners in trafficking. 2. Structure and function of the Bro1 domain in the ESCRT/monoubiquitin pathway Proteins delivered to the lysosome or the yeast vacuole via late endosomes are sorted by the ESCRT complexes and by associated proteins, including Alix and its yeast homolog Bro1. Alix, Bro1, and several other late endosomal proteins share a conserved 160 residue Bro1 domain whose boundaries, structure, and function have not been characterized. The crystal structure of the Bro1 domain of Bro1 reveals a folded core of 367 residues. The extended Bro1 domain is necessary and sufficient for binding to the ESCRT-III subunit Snf7 and for the recruitment of Bro1 to late endosomes. The structure resembles a boomerang with its concave face filled in, and contains a triple tetratricopeptide repeat domain as a substructure. Snf7 binds to a conserved hydrophobic patch on Bro1 that is required for protein complex formation and for the protein sorting function of Bro1. These results define a conserved mechanism whereby Bro1 domain-containing proteins are targeted to endosomes by Snf7 and its orthologs. Lipid and inositide signaling and trafficking 1. Activation of proteins by diacylglycerol The lipid second messenger diacylglycerol acts by binding to the C1 domains of target proteins, which translocate to cell membranes and are allosterically activated. Here we report the crystal structure at 3.2 ? resolution of one such protein, beta2-chimaerin, a GTPase activating protein for the small GTPase Rac, in its inactive conformation. The structure shows that in the inactive state, the N-terminus of beta2-chimaerin protrudes into the active site of the RacGAP domain, sterically blocking Rac binding. The diacylglycerol and phospholipid membrane binding site on the C1 domain is buried by contacts with the four different regions of beta2-chimaerin: the N-terminus, SH2 domain, RacGAP domain, and the linker between the SH2 and C1 domains. Phospholipid binding to the C1 domain triggers the cooperative dissociation of these interactions, allowing the N-terminus to move out of the active site and thereby activating the enzyme. 2. Structure of the catalytic core of inositol trisphosphate 5/6-kinase Inositol hexakisphosphate and other inositol high polyphosphates have diverse and critical roles in eukaryotic regulatory pathways. Inositol 1,3,4-trisphosphate 5/6-kinase catalyzes the rate-limiting step in inositol high polyphosphate synthesis in animals. This multifunctional enzyme also has inositol (3,4,5,6) tetrakisphosphate 1-kinase and other activities. The structure of an archetypal family member, from Entamoeba histolytica, has been determined to 1.2 ? resolution in binary and ternary complexes with nucleotide, substrate, and product. The structure reveals an ATP-grasp fold. The inositol ring faces ATP edge-on such that the 5 and 6-hydroxyl groups are nearly equidistant from the ATP ?-phosphate in catalytically productive phosphoacceptor positions and explains the unusual dual site specificity of this kinase. Inositol tris- and tetrakisphosphates interact via three phosphate binding subsites and one solvent-exposed site that could in principle be occupied by eighteen different substrates, explaining the mechanisms for the multiple specificities and catalytic activities of this enzyme. 3. Sterol transfer by OSBP family members The oxysterol binding protein (OSBP)-related proteins (ORPs) are conserved from yeast to man and are implicated in regulation of sterol pathways and in signal transduction. The structure of the full-length yeast ORP Osh4 was determined at 1.5-1.9 ? resolution in complexes with ergosterol, cholesterol, and 7-, 20-, and 25-hydroxycholesterol. A single sterol molecule binds in a hydrophobic tunnel in a manner consistent with a transport function for ORPs. The entrance is blocked by a flexible N-terminal lid and surrounded by functionally critical basic residues. The structure of the open state of a lid-truncated form of Osh4 was determined at 2.5 ? resolution. Structural analysis and limited proteolysis show that sterol binding closes the lid and stabilizes a conformation favoring transport across aqueous barriers and transmitting signals. The unliganded structure exposes potential phospholipid-binding sites that are positioned for membrane docking and sterol exchange. Based on these observations we propose a model in which sterol and membrane binding promote reciprocal conformational changes that facilitate a sterol transfer and signaling cycle.